Nies Anne T, Herrmann Elke, Brom Manuela, Keppler Dietrich
Division of Tumor Biochemistry, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
Naunyn Schmiedebergs Arch Pharmacol. 2008 Feb;376(6):449-61. doi: 10.1007/s00210-007-0219-x. Epub 2007 Dec 19.
An important function of hepatocytes is the biliary elimination of endogenous and xenobiotic small molecules, many of which are organic cations. To study this vectorial transport of organic cations, we constructed a double-transfected Madin-Darby canine kidney strain II (MDCKII) cell line permanently expressing the human organic cation transporter 1 (OCT1, SLC22A1) in the basolateral membrane and MDR1 P-glycoprotein (MDR1 P-gp, ABCB1), an adenosine triphosphate (ATP)-dependent efflux pump for organic cations, in the apical membrane. Additionally, MDCKII single transfectants stably expressing OCT1, MDR1 P-gp, or human organic cation transporter 2 (OCT2, SLC22A2) were generated. Antisera directed against OCT1 or OCT2 specifically detected OCT1 in the basolateral membrane of human hepatocytes, OCT2 in tubular epithelial cells of human kidney, and the respective recombinant transporter in the basolateral membrane of MDCKII transfectants. We identified the lipophilic organic cation berberine, a fluorescent plant alkaloid exhibiting a broad range of biological activities, as substrate of OCT1 and OCT2 with Michaelis-Menten constants of 14.8 microM and 4.4 microM, respectively. Berberine also inhibited the uptake of the prototypic cations tetraethylammonium and 1-methyl-4-phenylpyridinium by MDCK-OCT1 and MDCK-OCT2 transfectants. When transfected cells were grown polarized on permeable filter supports, berberine was transferred from the basolateral to the apical compartments many times faster by MDCK-OCT1/MDR1 P-gp double transfectants than by MDCK-OCT1 or MDCK-MDR1 P-gp single transfectants. The specific MDR1 P-gp inhibitor, zosuquidar trihydrochloride (LY335979), strongly inhibited berberine efflux into the apical compartment. The MDCK-OCT1/MDR1 P-gp double transfectants may be useful to identify additional cationic substrates and inhibitors of OCT1 and MDR1 P-gp, including drug candidates.
肝细胞的一项重要功能是通过胆汁消除内源性和外源性小分子，其中许多是有机阳离子。为了研究有机阳离子的这种向量转运，我们构建了一种双转染的马-达二氏犬肾II型（MDCKII）细胞系，该细胞系在基底外侧膜上永久表达人类有机阳离子转运体1（OCT1，SLC22A1），在顶膜上永久表达多药耐药蛋白1 P-糖蛋白（MDR1 P-gp，ABCB1），一种依赖三磷酸腺苷（ATP）的有机阳离子外排泵。此外，还生成了稳定表达OCT1、MDR1 P-gp或人类有机阳离子转运体2（OCT2，SLC22A2）的MDCKII单转染细胞。针对OCT1或OCT2的抗血清在人类肝细胞的基底外侧膜、人类肾脏的肾小管上皮细胞以及MDCKII转染细胞的基底外侧膜中特异性检测到了OCT1和各自的重组转运体。我们确定亲脂性有机阳离子黄连素（一种具有广泛生物活性的荧光植物生物碱）为OCT1和OCT2的底物，其米氏常数分别为14.8微摩尔和4.4微摩尔。黄连素还抑制了MDCK-OCT1和MDCK-OCT2转染细胞对原型阳离子四乙铵和1-甲基-4-苯基吡啶鎓的摄取。当转染细胞在可渗透滤膜支持物上极化生长时，MDCK-OCT1/MDR1 P-gp双转染细胞将黄连素从基底外侧转运到顶侧区室的速度比MDCK-OCT1或MDCK-MDR1 P-gp单转染细胞快许多倍。特异性MDR1 P-gp抑制剂三盐酸唑喹达（LY335979）强烈抑制黄连素向顶侧区室的外排。MDCK-OCT1/MDR1 P-gp双转染细胞可能有助于鉴定OCT1和MDR1 P-gp的其他阳离子底物和抑制剂，包括候选药物。
